FLOW CYTOMETRIC DETECTION OF HLA-SPECIFIC ANTIBODIES AS A PREDICTOR OF HEART ALLOGRAFT REJECTION1 (original) (raw)

Clinical Relevance of Anti-HLA Antibodies Detected by Flow-Cytometry Bead-Based Assays—Single-Center Experience

Human Immunology, 2006

The purpose of this study was to define the incidence, dynamics, and profiles of anti-human leukocyte antigen antibodies (HLA-Abs) produced after kidney transplantation and their impact on graft outcome. A total of 72 first cadaver donor kidney recipients were prospectively monitored for the development of HLA-Abs using beadbased flow-cytometry assays (One Lambda FlowPRA tests). Sixteen recipients (22.2%) developed HLA-Abs after transplantation (class I, n ϭ 7; class IϩII, n ϭ 6; class II, n ϭ 3), in most cases (81.25%) within the first 2 weeks posttransplantation. A strong association between alloantibody presence and delayed graft function (Chi-square ϭ 7.659, p Ͻ 0.01), acute rejection (Chi-square ϭ 14.504, p Ͻ 0.001), chronic rejection (Chi-square ϭ 12.84, p Ͻ 0.001), and graft loss (Chi-square ϭ 20.283, p Ͻ 0.001) was found. Patients with higher alloantibody titers experienced acute rejections and even early graft loss, compared with those with lower titers for whom chronic rejections were more common. Immunologic complications occurred in recipients with both donor-specific and cross-reacting groups or non-donor-specific antibodies alone. A positive correlation (Pearson correlation, 0.245; p Ͻ 0.05) between HLA class I amino acid triplet incompatibility and alloantibody production was observed, mainly resulting from immunogenic triplotypes. Given the results obtained in this study, an alloantibody testing algorithm has been designed and implemented for routine monitoring and to define optimally the alloantibody reactivity in kidney transplant recipients.

Outcome-Based Risk Assessment of Non-HLA Antibodies in Heart Transplantation: A Systematic Review

The Journal of Heart and Lung Transplantation, 2024

Background: Current monitoring after heart transplantation (HT) employs repeated invasive endomyocardial biopsies (EMB). Although positive EMB confirms rejection, EMB fails to predict impending, sub-clinical, or EMB-negative rejection events. While non-HLA antibodies have emerged as important risk factors for antibody-mediated rejection (AMR) after HT, their use in clinical risk stratification has been limited. A systematic review of the role of non-HLA antibodies in rejection pathologies has potential to guide efforts to overcome deficiencies of EMB in rejection monitoring. Methods: Databases were searched to include studies on non-HLA antibodies in HT recipients. Data collected included: number of patients, type of rejection, non-HLA antigen studied, association of non-HLA antibodies with rejection, and evidence for synergistic interaction between non-HLA antibodies and HLA-DSA responses. Results: A total of 56 studies met the inclusion criteria. Strength of evidence for each non-HLA antibody was evaluated based on the number of articles and patients in support vs. against their role in mediating rejection. Importantly, despite previous intense focus on the role of anti-MHC class I chain-related gene A (MICA) and anti-angiotensin II type I receptor (AT1R) antibodies in HT rejection, evidence for their involvement was equivocal. Conversely, strength of evidence for other non-HLA antibodies supports that differing rejection pathologies are driven by differing non-HLA antibodies. Conclusion: This systematic review underscores the importance of identifying peri-HT non-HLA antibodies. Current evidence supports the role of non-HLA antibodies in all forms of HT rejection. Further investigations are required to define the mechanisms of action of non-HLA antibodies in HT rejection.

Specificity and Sensitivity of Screening for Anti-HLA Antibodies in Kidney Allograft Dysfunction

Transplantation Proceedings, 2009

Background. Prospective testing for posttransplant circulating anti-HLA antibodies seems to be a critical noninvasive tool, but confirmatory data are lacking. Materials and Methods. Over the last 3 years, peritubular capillary (PTC) C4d deposition was prospectively sought by an immunofluorescence technique applied to frozen tissue in biopsies obtained for allograft dysfunction. Screening for circulating anti-HLA class I/II alloantibodies (AlloAb) by the flow cytometric test was performed simultaneously. Results. We evaluated 132 sets of biopsies and simultaneous serum samples. PTC C4d deposition was demonstrated in 15.9% (21/132) of biopsies. Circulating anti-HLA I/II AlloAb were detected in 25% (33/132) of serum samples. Employing receiver-operator characteristic (ROC) curves for all C4d-positive biopsies, screening for AlloAb showed a global specificity of 82% and sensitivity of 61.9%. When this analysis was restricted to biopsies obtained in the first month posttransplantation, the sensitivity increased to 81.8%, but the specificity decreased to 76.9%. After the first month posttransplantation, we observed sensitivity of 40.0% and a specificity of 86.4%. In the first month posttransplantation, all patients with a diagnosis of acute antibody-mediated rejection displayed circulating anti-HLA class I/II, but not always at the same time as the C4d-positive biopsy. Conclusions. In the first month posttransplantation, prospective monitoring of anti-HLA antibodies may be useful. The high sensitivity allows the identification of patients at risk, affording an earlier diagnosis of antibody-mediated rejection. After the first month, the test can be used to evaluate allograft dysfunction episodes, since positivity is highly suggestive of an antibody-mediated process.

Solid phase assays for HLA antibody detection in clinical transplantation

Current Opinion in Immunology, 2009

The complement dependent microlymphocytotoxicity assay has been used for over 40 years for detecting HLA antibodies in transplant patients. This method has been replaced recently by more sensitive solid phase assays such as ELISA and bead based technology including the Luminex method. The introduction of these techniques into clinical practice has revealed previously undetected sensitisation in some patients and allowed the accurate assignment of antibody specificities directed at HLA-DQ and HLA-DP which was not previously possible. However it is emerging that despite the advantage of sensitivity some HLA antibodies defined by these assays are not associated with hyperacute or acute rejection. The role in allograft rejection of antibody titre and non-complement fixing antibodies, which are also detected by these methods, are areas currently under consideration.

CLINICAL IMPLICATIONS OF THE MONITORING OF ANTI-HLA ANTIBODIES AFTER KIDNEY TRANSPLANTATION

Transplantation, 2008

The purpose of this study was to sequentially monitor anti-HLA antibodies and correlate the results with antibody-mediated rejection (AMR), graft survival (GS), and graft function (GF). We collected sera from 111 kidney transplant recipients on transplant days 0, 7, 14, 30, 60, 90, 180, and 360 and analyzed PRA levels by ELISA. DSAs were analyzed by single-antigen beads in rejecting kidneys. At pretransplant, 79.3% of the patients were non-sensitized (PRA = 0%) and 20.7% were sensitized (PRA > 1%). After transplant, patients were grouped by PRA profile: no anti-HLA antibodies pre-or post-transplant (group HLApreÀ/postÀ; n = 80); de novo anti-HLA antibodies posttransplant (group HLApreÀ/post+; n = 8); sensitized pre-transplant/ increased PRA post-transplant (group HLApre+/post↑; n = 9); and sensitized pre-transplant/decreased PRA post-transplant (group HLApre+/post↓; n = 14). De novo anti-HLA antibodies were detected at 7-180 d. In sensitized patients, PRA levels changed within the first 30 d post-transplant. Incidence of AMR was higher in HLApreÀ/post+ and HLApre+/post↑ than in HLApreÀ/postÀ, and HLApre+/post↓ (p < 0.001) groups. One-yr death-censored GS was 36% in group HLApre+/post↑, compared with 98%, 88% and 100% in groups HLApreÀ/postÀ, HLApreÀ/post+, and HLApre+/post↓, respectively (p < 0.001). Excluding first-year graft losses, GF and GS were similar among the groups. In conclusion, post-transplant antibody monitoring can identify recipients at higher risk of AMR.

Occurrence of Fatal and Nonfatal Adverse Outcomes after Heart Transplantation in Patients with Pretransplant Noncytotoxic HLA Antibodies

Journal of Transplantation, 2013

HLA antibodies (HLA ab) in transplant candidates have been associated with poor outcome. However, clinical relevance of noncytotoxic antibodies after heart transplant (HT) is controversial. By using a Luminex-based HLA screening, we retested pretransplant sera from HT recipients testing negative for cytotoxic HLA ab and for prospective crossmatch. Out of the 173 consecutive patients assayed (52 ± 13 ; 16% females; 47% ischemic etiology), 32 (18%) showed pretransplant HLA ab, and 12 (7%) tested positive against both class I and class II HLA. Recipients with any HLA ab had poorer survival than those without (65 ± 9 versus 82 ± 3%; = 0.02), accounting for a doubled independent mortality risk ( = 0.04). In addition, HLA-ab detection was associated with increased prevalence of early graft failure (35 versus 15%; = 0.05) and late cellular rejection (29 versus 11%; = 0.03). Of the subgroup of 37 patients suspected for antibody mediated rejection (AMR), the 9 with pretransplant HLA ab were more likely to display pathological AMR grade 2 ( = 0.04). By an inexpensive, luminex-based, HLA-screening assay, we were able to detect non-cytotoxic HLA ab predicting fatal and nonfatal adverse outcomes after heart transplant. Allocation strategies and desensitization protocols need to be developed and prospectively tested in these patients.

Clinical relevance of antibodies to HLA antigens undetectable by the standard complement-dependent cytotoxicity test

Transplant International, 2003

Abstract Recent literary data suggest that antibodies to HLA antigens undetectable by the standard complement-dependent cytotoxicity test may cause not only chronic, but also acute immunological complications after kidney transplantation. The aim of this study was to investigate the significance of non-cytotoxic antibodies to HLA antigens for the development of immunological complications and a worse graft prognosis after first kidney transplantation. Sera before and early after transplantation from 120 first kidney recipients were analyzed by flow cytometry (FCXM), ELISA and the standard complementdependent cytotoxicity (CDC) test. Pre-transplant FCXM negativity was related to a lower incidence of rejection episodes in the first posttransplant year (P<O.Ol). A significant association between acute rejection and the presence of antibodies to HLA class I1 antigens before and after transplantation was also found (P<O.O5). Our study supports the findings of other centers of the detrimental role to the kidney graft played by anti-HLA antibodies undetectable by the classical CDC test.

A novel strategy for the detection and definition of HLA-specific antibodies in patients awaiting renal transplantation

Transplant International, 1998

A novel strategy for the detection and definition of HLA-specific antibodies in patients awaiting renal transplantation Abstract Conventional testing for HLA-specific antibodies employs complement-dependent cytotoxicity (CDC) which is labour intensive and dependent on a supply of viable lymphocytes. Our strategy to minimise CDC screening is initially to screen sera by ELISA (Quikscreen) to detect HLA Class I-specific antibodies. Negative sera are then screened by flow cytometry (FCS) using lymphoblastoid cell line pools to detect HLA Class 11-specific antibodies. Only Quikscreen-or FCSpositive sera are then tested by CDC and, when indicated, with an ELISA kit (PRA-STAT) for specificity definition. Of 3680 sera, 886 (34.1 %) were Quikscreen positive. Of the 2794 Quikscreen-negative sera, 374 (13.4 %) were FCS positive. Therefore, only 1265 of the 3680 (34.3%) sera contained HLA-specific antibodies requiring specificity definition. This novel screening strategy has significantly reduced the CDC workload of the laboratory whilst enabling the detection of additional HLA-specific antibodies.